Mevalonate (MVA) metabolism provides the isoprenoids used in archaeal lipid biosynthesis. In synthesis of isopentenyl diphosphate, the classical MVA pathway involves decarboxylation of mevalonate diphosphate, while an alternate pathway has been proposed to involve decarboxylation of mevalonate monophosphate. To identify the enzymes responsible for metabolism of mevalonate 5-phosphate to isopentenyl diphosphate in Haloferax volcanii, two open reading frames (HVO_2762 and HVO_1412) were selected for expression and characterization. Characterization of these proteins indicated that one enzyme is an isopentenyl phosphate kinase that forms isopentenyl diphosphate (in a reaction analogous to that of Methanococcus jannaschii MJ0044). The second enzyme exhibits a decarboxylase activity that has never been directly attributed to this protein or any homologous protein. It catalyzes the synthesis of isopentenyl phosphate from mevalonate monophosphate, a reaction that has been proposed but never demonstrated by direct experimental proof, which is provided in this account. This enzyme, phosphomevalonate decarboxylase (PMD), exhibits strong inhibition by 6-fluoromevalonate monophosphate but negligible inhibition by 6-fluoromevalonate diphosphate (a potent inhibitor of the classical mevalonate pathway), reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that the PMD utilizes a reaction mechanism similar to that demonstrated for the classical MVA pathway decarboxylase. These observations represent the first experimental demonstration in H. volcanii of both the phosphomevalonate decarboxylase and isopentenyl phosphate kinase reactions that are required for an alternate mevalonate pathway in an archaeon. These results also represent, to our knowledge, the first identification and characterization of any phosphomevalonate decarboxylase.
Ribosomal protein L19 is an essential ribosomal protein and is a component of bridge B8, one of the protein-RNA bridges linking the large and small ribosomal subunits. Bridge B8 also contributes to the accuracy of translation by affecting GTPase activation by ribosome-bound aminoacyl tRNA-EF-Tu•GTP ternary complexes. Previous work has identified a limited number of accuracy-altering alterations in protein L19 of Salmonella enterica and Thermus thermophilus. Here, we have targeted the Escherichia coli rplS gene encoding L19 for mutagenesis and have screened for mutants with altered levels of miscoding. We have recovered 14 distinct L19 mutants, all of which promote increased stop codon readthrough, but do not have major effects on subunit association or cell growth. Examination of the E. coli 70S ribosome structure indicates that the amino acid substitutions cluster in three distinct regions of L19 and thereby potentially affect its interactions with L14 and 16S rRNA.
Enzymes of the isoprenoid biosynthetic pathway in halophilic archaea remain poorly characterized, and parts of the pathway remain cryptic. This situation may be explained, in part, by the difficulty of expressing active, functional recombinant forms of these enzymes. The use of newly available expression plasmids and hosts has allowed the expression and isolation of catalytically active Haloferax volcanii 3-hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (EC 2.3.310). This accomplishment has permitted studies that represent, to the best of our knowledge, the first characterization of an archaeal hydroxymethylglutaryl CoA synthase. Kinetic characterization indicates that, under optimal assay conditions, which include 4 M KCl, the enzyme exhibits catalytic efficiency and substrate saturation at metabolite levels comparable to those reported for the enzyme from nonhalophilic organisms. This enzyme is unique in that it is the first hydroxymethylglutaryl CoA synthase that is insensitive to feedback substrate inhibition by acetoacetyl-CoA. The enzyme supports reaction catalysis in the presence of various organic solvents. Haloferax 3-hydroxy-3-methylglutaryl CoA synthase is sensitive to inactivation by hymeglusin, a specific inhibitor known to affect prokaryotic and eukaryotic forms of the enzyme, with experimentally determined K i and k inact values of 570 ؎ 120 nM and 17 ؎ 3 min ؊1 , respectively. In in vivo experiments, hymeglusin blocks the propagation of H. volcanii cells, indicating the critical role that the mevalonate pathway plays in isoprenoid biosynthesis by these archaea
To identify the enzymes responsible for metabolism of mevalonate 5‐phosphate to isopentenyl diphosphate in H. volcanii, two open reading frames (HVO_2762; HVO_1412) were selected for expression and characterization. Characterization of these proteins indicates that one enzyme is an isopentenyl phosphate kinase that forms isopentenyl diphosphate. The second enzyme exhibits a decarboxylase activity that has never been directly attributed to this protein or any homologous protein. It catalyzes the synthesis of isopentenyl phosphate from mevalonate 5‐phosphate, a reaction proposed but never demonstrated by direct experimental proof. This enzyme, phosphomevalonate decarboxylase (PMD), exhibits strong inhibition by 6‐fluoromevalonate monophosphate but negligible inhibition by 6‐fluoromevalonate diphosphate (a potent inhibitor of the classical mevalonate pathway), reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that the PMD utilizes a reaction mechanism similar to that demonstrated for the classical MVA pathway decarboxylase. These observations represent the first experimental demonstration in H. volcanii of both the phosphomevalonate decarboxylase and isopentenyl phosphate kinase reactions that are required for an alternate mevalonate pathway in an archaeon. These results also represent, to the best of our knowledge, the first identification and characterization of an archaeal phosphomevalonate decarboxylase.
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